DESCRIPTION The long term goal of the proposed research is to understand the molecular mechanisms underlying motor nerve terminal differentiation. Regulated differentiation of nerve terminals is essential for the formation of appropriate connections in the developing nervous system. One important signal leading to terminal differentiation appears to come from the extracellular matrix protein agrin, previously known as an inducer of postsynaptic differentiation. Agrin can induce selective adhesion of motor neurons, inhibit outgrowth of motor neurites, and promote clustering of synaptic vesicles; these properties are expected of a presynaptic inducer. Inhibition of agrin function, in vivo or in vitro, leads to a failure of presynaptic differentiation. The proposed research is designed to answer several questions about agrin's role in presynaptic differentiation. First, how does agrin regulate motor neurons' growth and differentiation. The activities of neural vs. muscle agrin on motor neurons will be compared using transfected muscle cells and transgenic mice. Ca2+ signals induced in motor neurons by agrin will be characterized, and the relationship between these signals and agrin's activities will be examined. Second, what domains of agrin are required for interactions with neurons? Agrin isoforms and fragments will be used to determine the structural basis of agrin's actions on neurons. Finally, what are the neuronal receptors for agrin? The binding properties and localization of neuronal agrin binding proteins will be characterized, as a first step in the identification of functionally relevant neuronal agrin receptors. Additionally, the potential for various cell adhesion molecules to act as agrin receptors will be examined. The proposed experiments will provide key information concerning the mechanisms underlying nerve terminal formation by focusing on agrin, the single best candidate for a target-derived inducer of presynaptic differentiation. Information about the molecular basis of synaptogenesis may be useful in the diagnosis and treatment of developmental disorders of the brain and spinal cord. In addition, these results will bear on the mechanism of regeneration after nervous system injury.